Equilibrium and pressure-jump relaxation studies of the conformational transitions of P13MTCP1

The conformational transitions of a small oncogene product, p13(MTCP1), have been studied by high-pressure fluorescence of the intrinsic tryptophan emission and high-pressure 1D and 2D 1H-15N NMR. While the unfolding transition monitored by fluorescence is cooperative, two kinds of NMR spectral changes were observed, depending on the pressure range. Below approximately 200 MPa, pressure caused continuous, non-linear shifts of many of the 15N and 1H signals, suggesting the presence of an alternate folded conformer(s) in rapid equilibrium (tau<<ms) with the basic native structure. Above approximately 200 MPa, pressure caused a sharp decrease in the intensity of the folded proteins signals, while the peaks corresponding to disordered structures increased, yielding a free energy of unfolding change of 6.0 kcal/mol and associated volume change of -100 ml/mol, in agreement with the fluorescence result. Differential scanning calorimetry also reveals two transitions between 21 and 65 degrees C, confirming the existence of an additional species under mildly denaturing conditions. We report here a real-time observation of pressure-jump unfolding kinetics by 2D NMR spectroscopy on P13MTCP1 made possible due to its very long relaxation times at high pressure revealed by fluorescence studies. Within the dead-time after the pressure-jump, the NMR spectra of the native conformer changed to those of the transient conformational species, identified in the equilibrium studies, demonstrating the equivalence between a transient species and an equilibrium excited state. After these rapid spectral changes, the intensities of all of the individual 15N-1H cross-peaks decreased gradually, and those of the disordered structure increased, consistent with the slow relaxation to the unfolded form at this pressure. Rate constants of unfolding monitored at individual amide sites within the beta-barrel were similar to those obtained from fluorescence and from side-chain protons in the hydrophobic core region, consistent with nearly cooperative unfolding. However, some heterogeneity in the apparent unfolding rate constants is apparent across the sequence and can be understood as non-uniform effects of pressure on the unfolding rate constant due to non-uniform hydration.